Today the biofuel industry primarily produces ethanol from traditional feedstocks such as cereal crops like corn and wheat and sugar crops like sugar cane and sweet sugar beet.
The fermentation of plant carbohydrates by yeast dates back to the Neolithic period [1]. Today, yeasts are widely used in many biotechnological processes, with the largest use for the production of alcoholic beverages such as beer and wine and fuel ethanol. The yeast Saccharomyces cerevisiae is the predominant microorganism for industrial ethanol production. This yeast is characterized by several desirable industrial properties which include fast growth, efficient glucose anaerobic metabolism, high ethanol productivity and yield, and tolerance to several environmental stress-factors, such as high ethanol concentration and low oxygen level [2]. In spite of good ethanol yields from glucose and sucrose, the yield from these sugars can be further increased. There are several approaches that can be employed for this, which include genetic engineering techniques that target the redirection of yeast metabolism towards increased yield of ethanol production from carbohydrates by reducing co-product generation [3]. Particularly, this can be achieved through the reduction of glycerol and biomass synthesis, since these represent the major by-products made during a bioethanol production. During anaerobic growth of S. cerevisiae, glycerol serves as an essential electron sink for re-oxidizing reduced NADH generated during biosynthesis of ethanol. The employment of engineering approaches, which aim to change cellular redox metabolism via reduced formation of cytosolic NADH, resulted in the reduction of glycerol production with increase in ethanol yield [4, 5, 6, 7]. Decreasing biomass accumulation is an alternative way to increase ethanol yield from sugar consumed. The extent of biomass accumulation is dependent on the availability of energy in the form of ATP. If cellular ATP yield is decreased, it was anticipated that an increase in ethanol yield will result [8]. As predicted, increase in ethanol production was demonstrated using metabolic engineering approaches via the introduction of ATP-hydrolyzing futile cycles in yeast through the deregulation of some of the gluconeogenic enzymes [9]. A positive effect on ethanol production was also accomplished by the overexpression of ATPase [10] or the alkaline phosphatase Pho8 which can also hydrolyze ATP [11].
Though redirecting yeast metabolism towards increasing ethanol production using genetic engineering approaches is relatively easy to achieve, the usage of genetically modified strains for ethanol production, mainly in wine yeast, is not accepted in many parts of the world due to consumer's resistance to the use of genetically modified organisms (GMO) for beverage alcohol [12]. For this reason, the use of non-GMO approaches, which consist of traditional selection and adaptive evolution, must be relied on developing new improved strains [13]. Traditional selection and adaptive evolution involve applying culture conditions that provide a selection pressure favoring the growth of mutants within a population that confer the trait of interest. Thus, the culturing of cell populations in a specific selective environment will direct the accumulation of adaptation towards a desired phenotype [13, 14, 15]. There are several excellent examples that illustrate the usefulness of this approach to the selection of anaerobical xylose utilizing yeast strains [16], yeast with enhanced maltose utilization and osmotolerance [17], yeast with enhanced ethanol tolerance [18] and yeast with improved fermentation rate with decreased formation of acetate [19].
In the present disclosure, there is described new methods for positive selection of S. cerevisiae strains with enhanced ethanol production phenotypes. The methods are exemplified using industrial S. cerevisiae AS400 strain that is used for bioethanol production at Archer Daniels Midland Company (Decatur, Ill., USA). The selective agents oxythiamine, trehalose, 3-bromopyruvate, glyoxylic acid and glucosamine, were used in this description and chosen for their inhibitory effect on the enzymes involved in alcoholic fermentation and stress response.